anti-A IgG Research Articles

Relative Quantification for Obtaining the IgG Blood Group Antibodies in Plasma by the Absorption and Elution Test

To establish the absorption and elution test for relatively quantitive obtaining anti-A and anti-B blood group IgG antibodies in the plasma of O-type RhD-positive pregnant women. 95 cases of the O-type RhD-positive pregnant women plasma samples were randomly selected for obtaining the IgG antibodies of anti-A and anti-B blood group, with absorption test under 37 ℃ and elution test under 56 ℃, and the IgG anti-A and anti-B antibody titers of plasma and elution were determined by the microcolumn gel anti-human globulin test. The differences and correlation between the titers of IgG antibodies in the eluent and plasma were compared and analyzed. After a logarithmic transformation (Log2), there was no statistically difference between IgG antibody anti-A difference value and anti-B difference value in the eluent and plasma (P >0.05). The titer of IgG antibody in the eluent was positively correlated with the titer of IgG antibody in the plasma (r =0.914). The linear equation for IgG antibody titers fitted by a scatter plot between the eluent and plasma was Y=-3.55+0.96X. The absorption and elution test can be used to obtain the anti-A and anti-B IgG antibodies in the plasma of O-type RhD-positive pregnant women, whose plasma origin IgG titer is greater than 8. Meanwhile, the acquisition of anti-A antibodies was as effective as anti-B antibodies at the same time, and the antibodies obtained are positive proportional to their respective concentrations in the plasma.

An in vitro comparison of intra-operative isohemagglutinin and human leukocyte antigen removal techniques in pediatric heart transplantation

Background: Highly sensitized pediatric patients awaiting heart transplantation experience longer wait times and thus higher waitlist mortality. Similarly, children less than 2 years of age have increased waitlist times and mortality when compared to their older peers. To improve the likelihood of successful transplantation in these patients, various strategies have been utilized, including peri-operative plasmapheresis. However, limited data exists comparing plasmapheresis techniques for antibody reduction. This study’s aim was to compare the in vitro magnitude of isohemagglutinin titers (IT) and human leukocyte antigen (HLA) antibody removal and the time required between membrane-based plasmapheresis (MP) and centrifuge-based plasmapheresis (CP) incorporated into the extracorporeal (EC) circuit. Methods: Two MP (Prismaflex) and two CP (Spectra Optia, Terumo BCT) circuits were incorporated into four separate EC circuits primed with high titer, highly sensitized type O donor whole blood. Assays were performed to determine baseline IT and anti-HLA antibodies and then at 30-minute increments until completion of the run (two plasma volume exchanges) at two hours. Results: There was a decrease in anti-A and anti-B IgM and IgG titers with both MP and CP. Mean anti-A and anti-B titer reduction was by 4.625 titers (93.7% change) and 4.375 titers (93.8% change) using MP and CP, respectively. At 2 h of apheresis, CP reduced 62.5% of all ITs to ≤ 1:4, while MP reduced 50% of ITs to ≤ 1:4. Additionally, reduction of anti-HLA class II antibody to mean fluorescence intensity (MFI) <3000 was achieved with both MP and CP. At 2 h of apheresis, CP reduced MFI by 2–3.5 fold and MP reduced MFI by 1.7–2.5 fold. Both demonstrated similar hemolytic and thrombotic profiles. Conclusions: In this in vitro plasmapheresis model of IT and anti-HLA antibody reduction, both MP and CP incorporated into the EC circuit can be used quickly and effectively to reduce circulating antibodies. While CP may have some greater efficiency, further study is necessary to verify this in vivo.

An optimized microplate-based method to evaluate complement-dependent hemolysis mediated by intravenous immunoglobulins (IVIG)

Hemolytic reactions can cause serious complications after administration of Intravenous Immunoglobulin (IVIG), due to passive transfer of anti-A and anti-B IgG antibodies (isoagglutinins). A maximum allowable amount of isoagglutinins is established in the US and EU for licensed IVIG, as measured by a specified direct hemagglutination test (DHAT). Despite this limit, reports of hemolysis have increased over time, raising the question of how well the DHAT predicts clinically significant hemolysis. This study was undertaken to develop a microplate-based complement-dependent hemolysis assay (CDHA) that reproducibly measures functional hemolytic activity of IVIG, for assessment of IVIG products. An IVIG working reference reagent (NIBSC 14/160) was qualified as an assay control and for quantitation purposes. Hemolytic activities of 36 IVIG product lots encompassing seven brands and including 6 clinically hemolytic lots were measured. Hemolytic activity varied among IVIG product brands, and to a lesser extent, from lot-to-lot for individual brands. Correlation between the CDHA and DHAT was not robust which may reflect imprecision of the DHAT method or additional variables that influence complement-dependent hemolysis after opsonization. In conclusion, the CDHA provides a simple, specific, and sensitive tool for IVIG product characterization and investigation of hemolytic events by manufacturers, researchers, and regulatory authorities.

P1387: MONITORING OF DONOR CHIMERISM ON RED BLOOD CELLS ANTIGENS AFTER HEMATOPOIETIC STEM CELL TRANSPLANTATION

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is an effective treatment for diseases of the blood system. One of the indicators reflecting the functioning of donor hematopoietic tissue is posttransplant chimerism. Aims: To evaluate the effect of ABO belonging of the donor and recipient on the timing of the formation of red blood cells (RBS) donor chimerism after HSCT. Methods: The study included 57 patients who underwent HSCT in 2013-2021. Patient age ranged from 3 to 60 years (median - 34). Diagnosis of the patients: acute leukemia - 42 persons, Hodgkin’s lymphoma - 6, aplastic anemia - 4 and other blood diseases - 5. Before HSCT, the study of RBC antigens of the ABO, Rh, Kell systems in donors and recipients was performed by gel agglutination using reagents and equipment BioRad (USA). Antigens identified in the recipient’s phenotype and absent in the donor’s phenotype were considered informative, or vice versa. For statistical data processing, the nonparametric Kruskal-Wallis test was used. Results: Thirty-two (56.1%) of 57 pairs differed in ABO system antigens. Of these, 16 pairs (28.1%) had a major ABO incompatibility, 16 pairs (28.1%) had a minor incompatibility. The time of appearance of donor’s RBS in patients with ABO incompatibility had a wider range (from 14 to 201 days) and occurred significantly later (Me - 28 days) than in recipients ABO identical with donors (Me - 20 days), and from minor ABO incompatibility (Me - 19 days) (Table 1). The periods of occurrence of 50% and 100% donor chimerism did not differ significantly in all groups. In the group of recipients with major ABO incompatibility a case of pure red cell aplasia was revealed. The first appearance of RBC carrying donor antigens was recorded on day 201. 100% donor chimerism was formed on day 278. Antibodies of the ABO system (anti-A) IgG were present in the recipient’s blood for 189 days, IgM - for 230 days. Table 1. - The emergence of chimerism depending on ABO compatibility of donor-recipient pairs ABO identical Minor ABO incompatibility Major ABO incompatibility р (n=25) (n=16) (n=16) First appearance of donor chimerism 20 (12-54) 19 (11-41) 28 (14-201) р<0.05 Ме (Min-Max days) 50% donor chimerism 41 (19-142) 41 (26-74) 54 (34-227) p>0.05 Ме (Min-Max days) 100% donor chimerism 138 (40-316) 110 (70-150) 141 (90-278) p>0.05 Ме (Min-Max days) Summary/Conclusion: In recipients with a major ABO incompatibility the first appearance of donor’s RBS after HSCT occurs significantly later than in couples ABO identical and couples with a minor ABO incompatibility.

MP36-05 A NOVEL METHOD OF CD31 COMBINED ABO CARBOHYDRATE ANTIGEN MICROARRAY PREDICTS ACUTE ANTIBODY MEDIATED REJECTION IN ABO-INCOMPATIBLE KIDNEY TRANSPLANTATION

You have accessJournal of UrologyCME1 May 2022MP36-05 A NOVEL METHOD OF CD31 COMBINED ABO CARBOHYDRATE ANTIGEN MICROARRAY PREDICTS ACUTE ANTIBODY MEDIATED REJECTION IN ABO-INCOMPATIBLE KIDNEY TRANSPLANTATION Masayuki Tasaki, Hiroaki Tateno, Takashi Sato, Azusa Tomioka, Hiroyuki Kaji, Hisashi Narimatsu, Kazuhide Saito, Toshinari Aoki, Masami Kamimura, Takashi Ushiki, Yutaka Yoshida, Kota Takahashi, and Yoshihiko Tomita Masayuki TasakiMasayuki Tasaki More articles by this author , Hiroaki TatenoHiroaki Tateno More articles by this author , Takashi SatoTakashi Sato More articles by this author , Azusa TomiokaAzusa Tomioka More articles by this author , Hiroyuki KajiHiroyuki Kaji More articles by this author , Hisashi NarimatsuHisashi Narimatsu More articles by this author , Kazuhide SaitoKazuhide Saito More articles by this author , Toshinari AokiToshinari Aoki More articles by this author , Masami KamimuraMasami Kamimura More articles by this author , Takashi UshikiTakashi Ushiki More articles by this author , Yutaka YoshidaYutaka Yoshida More articles by this author , Kota TakahashiKota Takahashi More articles by this author , and Yoshihiko TomitaYoshihiko Tomita More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002590.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Isohemagglutinin assays employing red blood cells (RBCs) are the most common assays used to measure antibody titer in ABO-incompatible kidney transplantation (ABOi KTx). However, ABO blood group antigens expressed on RBCs are not identical to those of the kidney due to different proteins linked to ABO carbohydrate antigens. Antibody titers measured by isohemagglutinin assays do not always correlate with clinical outcome and a new method is necessary to precisely predict acute antibody mediated rejection (ABMR) following ABOi KTx. METHODS: We previously reported that CD31 was the most abundant protein linked to ABO carbohydrate antigens on kidney endothelial cells, and developed a new method to measure antibody titer using a microarray of recombinant CD31 (rCD31) linked to ABO carbohydrate antigens (CD31-ABO microarray. Figure 1). To confirm clinical use, a total of 252 plasma samples including volunteers, hemodialysis patients and transplant recipients were examined by both of CD31-ABO microarray and isohemagglutinin assays. RESULTS: Mass spectrometry analysis suggested that rCD31 and native CD31 had similar ABO glycan, meaning CD31-ABO microarray were the mimic of ABO antigens on renal endothelial cells. In transplant recipients, any initial IgG or IgM antibody intensity >30,000 against the donor blood type in the CD31-ABO microarray showed higher sensitivity, specificity, positive predictive value, and negative predictive value to predict acute ABMR following ABOi KTx, compared to isohemagglutinin assays. Representative data were shown in Figure 2 (Blood group A-incompatible). Ten out of 12 patients with ABMR (83.3%) had anti-A IgG Ab levels >30,000 in the CD31-ABO microarray, in contrast, only 1 out of 17 patients without ABMR (5.9%) had anti-A IgG Ab levels >30,000 (Figure 5a). No one had anti-A IgM Ab levels >30,000 in the ABMR (-) group (Figure 5b). CONCLUSIONS: Use of a CD31-ABO microarray will contribute to precisely predicting ABMR following ABOi KTx in advance. Source of Funding: This study was supported by JSPS KAKENHI Grant Number 17K11195 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e598 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Masayuki Tasaki More articles by this author Hiroaki Tateno More articles by this author Takashi Sato More articles by this author Azusa Tomioka More articles by this author Hiroyuki Kaji More articles by this author Hisashi Narimatsu More articles by this author Kazuhide Saito More articles by this author Toshinari Aoki More articles by this author Masami Kamimura More articles by this author Takashi Ushiki More articles by this author Yutaka Yoshida More articles by this author Kota Takahashi More articles by this author Yoshihiko Tomita More articles by this author Expand All Advertisement PDF DownloadLoading ...

High titers of anti-A1 and anti-B antibodies among Peruvian group O platelet donors

Critical antibody titers have been described as factors associated with hemolysis in ABO plasma-incompatible platelet (PLT) transfusions. This study was carried out to describe the frequency of high-titers anti-A and antiB IgM and IgG antibodies in group O apheresis platelet donors, and to explore differences according to the donor characteristics. A cross-sectional study was carried out at the Blood Bank of a National Hospital in Peru from January to March 2019. IgM and IgG antibodies against A1 and B antigens were quantified in 339 platelet donors using the direct hemagglutination technique and the solid-phase adherence technique, respectively. For analysis purposes, two cut-off points; ≥128 and ≥64, were used to define a critical titer for IgM due to a lack of consensus. An IgG titer of ≥256 was also defined as critical. Of the donors, 22.1 % had critical IgM titers when the cut-off point was defined as ≥128. However, when the IgM cut-off was ≥64, the frequency of platelet donors with critical titers increased to 54.0 %. The frequency of donors with critical IgG titers was 23.5 %. Higher IgG titers were associated with female donors while higher IgM titers were negative associated with age. One in two or three platelet donors, depending on the cutoff point used to define a critical IgM titer, had at least one critical titer of anti-A or anti-B antibodies. Early identification of platelet donors with critical antibody titers could prevent passive transfusion of ABO antibodies to non-isogroup recipients.

Development of a Complement-Mediated Hemolysis Assay for Immune Globulin Intravenous Products and Characterization of Lots Associated with Clinical Hemolysis

Introduction: Immune Globulin Intravenous (Human) (IGIV), as known as Intravenous Gamma-Globulin (IVIg), are therapeutic polyclonal immunoglobulin preparations manufactured from pooled human plasma. Initially licensed for replacement therapy of primary immune deficiencies, IGIV indications have expanded to include various autoimmune and inflammatory conditions. Hemolytic reactions associated with IGIV infusion were historically considered as uncommon, but seem to be more frequent over the past 5-6 years, coinciding with widespread manufacturing changes to increase IgG yield, and use of higher dosages. Anti-A and anti-B IgG isoagglutinins in the IGIV preparations are believed to mediate these events. In 2014, FDA co-organized a workshop to discuss strategies to address hemolytic complications of IGIV infusions (Scott 2015). Although US products all comply with an anti-A and anti-B specification of ≤1:64, using the European Pharmacopoeia direct hemagglutination test (DHAT) (Thorp 2010), hemolytic events have persisted. The DHAT is semi-quantitative, imprecise, and does not measure hemolytic function. However, hemolysin tests have been difficult to standardize, especially for highly concentrated IGIV products (Scott 2015).This study reports development of a complement-dependent hemolysis assay (CDHA) suitable for testing IGIV products. We also tested a positive control preparation that is available from NIBSC (code 14/160), consisting of an IGIV lot that caused clinical hemolysis. Finally, we demonstrated differences among IGIV brands with respect to hemolytic activity.Methods: To provide consistent reagents, serum from whole blood donations were frozen as a large volume source of complement and a droplet-freezing method was used to store Red Blood Cells (Schmid 2011; Figure 1). Critical assay conditions were studied, including enzymatic treatment of RBC, removal of unbound IgG, complement source and concentration, divalent cation concentrations, and pH. Hemolytic activities of IGIV products and a positive control IGIV preparation were compared to the hemagglutination results tested using DHAT method. The CDHA was also used to evaluate hemolytic activity of IgG subclasses.Results: The most critical parameters for reproducible CDHA were complement source and preparation method, and assay pH. A pH of 6.5 (0.1M Tri-BIS buffer) was the most suitable condition for all of the tested IGIV analytes. Intra-laboratory variation was minimized by use of frozen RBCs and a consistent highly active complement preparation. The DHAT and CDHA results correlated with each other for most IGIV product lots (Figure 2). Hemolytic activity in IGIV lots varied among, in some cases within, brands. Enrichment of IgG subclass fractions from NIBSC 14/160 showed the highest hemolytic activity was present in IgG2 (Figure 3). In contrast, studies of recombinant anti-A monoclonal antibodies expressing the same anti-A F(ab'), but subclass-specific Fc receptors showed greatest activity of IgG3, consistent with evidence that IgG3 is the most potent complement-activating subclass. The role of IgG2 in hemolysis is not entirely surprising, as saccharide antigens often elicit IgG2 responses, and IgG2 can mediate complement activation (Diebolder 2014). Further subclass studies are planned.Conclusion: Hemolytic activity of IGIV products mediated by anti-A isoagglutinins can be reproducibly measured in IGIV products (Figure 4). Compared to the DHAT method, CDHA provides a continuous readout, is less variable, amenable to use of a standard curve, and more precise. Historical challenges to reproducibility are addressed through biological reagent standardization and attention to critical conditions. Studies comparing the CDHA and DHAT methods across a wider range of products and hemolytic lots will discern whether the CDHA provides a better measure of product safety than the current DHAT lot release assay. The CDHA can be helpful for characterization of new manufacturing methods and products, and investigation of hemolytic events. Studies of hemolysis-implicated lots may provide a basis for improvements in product safety. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Complement activating ABO anti-A IgM/IgG act synergistically to cause erythrophagocytosis: implications among minor ABO incompatible transfusions

BackgroundTypically minor ABO incompatible platelet products are transfused without any incident, yet serious hemolytic transfusion reactions occur. To mitigate these events, ABO ‘low titer’ products are used for minor ABO incompatible transfusions. We sought to understand the role of IgM/IgG and complement activation by anti-A on extravascular hemolysis.MethodsSamples evaluated included (i) Group O plasma from a blood donor whose apheresis platelet product resulted in an extravascular transfusion reaction, (ii) Group O plasma from 12 healthy donors with matching titers that activated complement (N = 6) or not (N = 6), and (iii) Group O sera from 10 patients with anti-A hemolysin activity. A flow cytometric monocyte erythrophagocytosis assay was developed using monocytes isolated by immunomagnetic CD14-positive selection from ACD whole blood of healthy donors. Monocytes were frozen at − 80 °C in 10% dimethyl sulfoxide/FBS and then thawed/reconstituted on the day of use. Monocytes were co-incubated with anti-A-sensitized fluorescently-labeled Group A1 + RBCs with and without fresh Group A serum as a source of complement C3, and erythrophagocytosis was analyzed by flow cytometry. The dependency of IgM/IgG anti-A and complement C3 activation for RBC erythrophagocytosis was studied. Anti-A IgG subclass specificities were examined for specific samples.ResultsThe plasma and sera had variable direct agglutinating (IgM) and indirect (IgG) titers. None of 12 selected samples showed monocyte-dependent erythrophagocytosis with or without complement activation. The donor sample causing a hemolytic transfusion reaction and 2 of the 10 patient sera with hemolysin activity showed significant erythrophagocytosis (> 10%) only when complement C3 was activated. The single donor plasma and two sera demonstrating significant erythrophagocytosis had high IgM (≥ 128) and IgG titers (> 1024). The donor plasma anti-A was IgG1, while the patient sera were an IgG3 and an IgG1 plus IgG2.ConclusionHigh anti-A IgM/IgG titers act synergistically to cause significant monocyte erythrophagocytosis by activating complement C3, thus engaging both Fcγ- and CR1-receptors.

A Comparison of Intra-Operative Isohemagglutinin Removal Techniques in Pediatric Heart Transplantation

ABO-incompatible (ABO-I) heart transplantation (HT) is used in children <2 years of age to reduce waitlist times and mortality with outcomes comparable to ABO-compatible HT. This strategy is made safer by reducing isohemagglutinin titers (IT). Limited data exists comparing techniques for IT reduction. This study's aim was to compare the in vitro magnitude of IT removal and time required between membrane-based plasmapheresis (MP) and centrifuge-based plasmapheresis (CP) incorporated into the cardiopulmonary bypass (CPB) circuit. Two MP (Prismaflex) and two CP (Spectra Optia, Terumo BCT) circuits were incorporated into four separate CBP circuits primed with high titer type O donor whole blood. Assays were performed to determine baseline isohemagglutinin titers and then at 30 minute increments until completion of the run (two plasma volume exchanges) at two hours. There was a decrease in anti-A and anti-B IgM and IgG titers with both MP and CP. Mean anti-A and anti-B titer reduction was 4.625 titers (93.7% change) and 4.375 titers (93.8% change) using MP and CP, respectively. At 2 hours of apheresis, CP reduced 62.5% of all ITs to ≤ 1:4, while MP reduced 50% of ITs to ≤1:4. Both demonstrated similar hemolytic and thrombotic profiles. In this in vitro plasmapheresis model of IT reduction, both MP and CP incorporated into the CPB circuit can be used quickly and effectively to reduce circulating IT. While CP may have some greater efficiency, further study is necessary to verify this in vivo.

A Case Report: First Ever ABO Incompatible Kidney Transplantation in Bangladesh: The Start of a New Era

Although ABO incompatible kidney transplantations are increasingly being practised worldwide, in Bangladesh it was never attempted. Here we report the first ever ABO incompatible kidney transplantation case performed in Bangladesh. The patient was a 23-year-old male with end-stage kidney disease secondary to chronic glomerulonephritis on haemodialysis. The only kidney donor available was his mother who had a different blood-group (patient O positive; mother A positive). His baseline anti-A IgG titre was 1:128. We used a modified desensitization protocol with low-dose Rituximab, triple immunosuppression (Tacrolimus, Mycophenolate Sodium and Prednisolone) and 3 sessions of plasmapheresis followed by IVIG. His Anti-A IgG titre was brought down to 1: 8 before transplantation. He also received basiliximab as induction therapy. He successfully underwent renal transplantation across the ABO barrier. After transplantation his IgG titre was monitored which was remained 1:4. At 1, 3 and 6 months of post transplant, serum creatinine level was 207 µmol/L, 182 µmol/L and 183 µmol/L, respectively. This was the first case of ABO-incompatible kidney transplantation in Bangladesh. This case gives us the confidence that more ABO-incompatible kidney transplantations can be performed in a resource limited setting and high infection risk countries like ours following a more cautious approach. This will substantially increase the living kidney donor pool; hence reduce the economic and health impact of continuing long term dialysis therapy in Bangladesh.

Next Generation Sequencing-Based Fetal ABO Blood Group Prediction by Analysis of Cell-Free DNA from Maternal Plasma

Introduction: ABO blood group incompatibility between a pregnant woman and her fetus as a cause of morbidity or mortality of the fetus or newborn remains an important, albeit rare, risk. When a pregnant woman has a high level of anti-A or anti-B IgG antibodies, the child may be at risk for hemolytic disease of the fetus and newborn (HDFN). Performing a direct prenatal determination of the fetal ABO blood group can provide valuable clinical information. Objective: Here, we report a next generation sequencing (NGS)-based assay for predicting the prenatal ABO blood group. Materials and Methods: A total of 26 plasma samples from 26 pregnant women were tested from gestational weeks 12 to 35. Of these samples, 20 were clinical samples and 6 were test samples. Extracted cell-free DNA was PCR-amplified using 2 primer sets followed by NGS. NGS data were analyzed by 2 different methods, FASTQ analysis and a grep search, to ensure robust results. The fetal ABO prediction was compared with the known serological infant ABO type, which was available for 19 samples. Results: There was concordance for 19 of 19 predictable samples where the phenotype information was available and when the analysis was done by the 2 methods. For immunized pregnant women (n = 20), the risk of HDFN was predicted for 12 fetuses, and no risk was predicted for 7 fetuses; one result of the clinical samples was indeterminable. Cloning and sequencing revealed a novel variant harboring the same single nucleotide variations as ABO*O.01.24 with an additional c.220C>T substitution. An additional indeterminable result was found among the 6 test samples and was caused by maternal heterozygosity. The 2 indeterminable samples demonstrated limitations to the assay due to hybrid ABO genes or maternal heterozygosity. Conclusions: We pioneered an NGS-based fetal ABO prediction assay based on a cell-free DNA analysis from maternal plasma and demonstrated its application in a small number of samples. Based on the calculations of variant frequencies and ABO*O.01/ABO*O.02 heterozygote frequency, we estimate that we can assign a reliable fetal ABO type in approximately 95% of the forthcoming clinical samples of type O pregnant women. Despite the vast genetic variations underlying the ABO blood groups, many variants are rare, and prenatal ABO prediction is possible and adds valuable early information for the prevention of ABO HDFN.

Evaluation of anti-A and anti-B IgM and anti-AB IgG antibody titers in Group O blood Iraqi donors

BACKGROUND: Blood group O is transfused as a universal blood group in emergencies. The red cells of blood Group O possess no major ABO antigens, while the blood Group O plasma contains naturally occurring IgM anti-A and anti-B and cross-reacting IgG anti-AB antibodies. Thus, the transfusion of blood Group O units may cause marked destruction of the recipients' A or B red cells manifested as severe acute hemolytic transfusion reactions owing to potent ABO hemolytic antibodies.OBJECTIVES: This study was done to estimate the titers of anti-ABO antibodies in Group O blood donors and to suggest a possible scheme to introduce as a routine testing for high ABO antibodies' titers in blood Group O donors in Iraqi blood transfusion centers.SUBJECTS AND METHODS: Two hundred blood Group O Iraqi healthy blood donors (192 males and 8 females, age range: 18–59) were chosen to evaluate the titer of ABO antibodies (IgM and IgG) in their serums using tube test.RESULTS: The prevalence of high-titer anti-A and anti-B IgM antibody at 1:50 dilution in this study was 98.5% and 97.5%, respectively (P < 0.05). The prevalence of high-titer anti-AB IgG measured by dithiothreitol and 2-mercaptoethanol method of at least 50 was 63% and 74%, respectively (P < 0.05).CONCLUSIONS: There is a high prevalence of high-titer anti-A and anti-B IgM and IgG antibodies among blood Group O Iraqi donors. The detection of IgG antibodies is more practical by using dithiothreitol method as it needs much shorter incubation time; however, 2-ME is more sensitive. Detection of high-titer anti-A IgM antibody in dangerous universal donors, by immediate spin at 1:50 dilution, can be used alone in practice, replacing the need to use all portfolio of tests of anti-ABO antibodies.

Prevalence of high titered anti-A and anti-B antibodies among O blood group individuals and their associated factors

Background and Objectives: Severe hemolytic transfusion reactions due to minor incompatibility are usually associated with the so-called “High titred” antibodies in the plasma of donors especially the O group as it is generally regarded as universal donors. Critical titers of 1:64 or higher was considered high titered antibodies for IgM anti-A and anti-B. Critical titers of 1:256 or higher was considered high titered antibodies for IgG anti-A and anti-B. The objective of this study was to see the prevalence of such high titered antibodies among our donor population and to observe for an association if any with the age, gender, and diet type and probiotic intake of the donors. Methodology: This was a cross-sectional analytical study conducted over 20 months from September 2015 to May 2017. About 560 donors were chosen randomly after screening and counseling and titration of anti-A and anti-B antibodies was done by the double dilution tube agglutination technique. The information on age, gender, diet type, and probiotic intake was gathered from the donor history questionnaire and an association between the factors described and the distribution of critical titers of anti-A and anti-B was elicited. Results: In the present study, the prevalence of high titer anti-A and anti-B IgM antibodies was 36% and 32%, respectively. The prevalence of high titer anti-A and anti-B IgG antibodies was 9.6% and 5.5%, respectively. The median IgM antibody titers reduced with the increasing age of the donors starting 18 years. There was a statistically significant association between a vegetarian diet and high titers. Conclusion: There are a considerable proportion of O group donors with high titers of antibodies and hence a routine pretransfusion screening for such antibodies can prevent the development of hemolytic reactions by segregating units into “high titer” and “low titer” antibodies and issuing only low titer products for out of group transfusions.

A2 to B Kidney Transplantation in the Post-Kidney Allocation System Era: A 3-year Experience with Anti-A Titers, Outcomes, and Cost

The new kidney allocation systems (KAS) instituted December 2014 permitted A2 to B deceased donor kidney transplantation (DDKTx) to improve access and reduce disparities in wait time for minorities. A recent United Network for Organ Sharing (UNOS) analysis, however, indicated only 4.5% of B candidates were registered for A2 kidneys. Cited barriers to A2 to B DDKTx include titer thresholds, patient eligibility, and increased costs. There are little published data on post-transplantation anti-A titers or outcomes of A2 to B DDKTx since this allocation change. We conducted a retrospective, single center, cohort analysis of 29 consecutive A2 to B and 50B to B DDKTx from December 2014 to December 2017. Pre- and postoperative anti-A titers were monitored prospectively. Outcomes included post-transplant anti-A titers, patient and graft survival, renal function, and hospital costs. African Americans comprised 72% of the A2 to B and 60% of the B to B group. There was no difference in mean wait time (58.8 vs 70.8 months). Paired tests indicated that anti-A IgG titers in A2 to B DDKTx were increased at discharge (p= 0.001) and at 4 weeks (p= 0.037). There were no significant differences in patient or graft survival, serum creatinine (SCr), or estimated glomerular filtration rate (eGFR), but the trajectories of SCr and eGFR differed between groups over the follow-up period. A2 to B had significantly higher mean transplant total hospital costs ($114,638 vs $91,697, p < 0.001) and hospital costs net organ acquisition costs ($42,356 vs $20,983, p < 0.001). Initial experience under KAS shows comparable outcomes for A2 to B vs B to B DDKTx. Anti-A titers increased significantly post-transplantation, but did not adversely affect outcomes. Hospital costs were significantly higher with A2 to B DDKTx. Transplant programs, regulators, and payors will need to weigh improved access for minorities with increased costs.

Long-term desensitization for ABO-incompatible living related kidney transplantation recipients with high refractory and rebound anti-blood type antibody: case report

BackgroundABO-incompatible living related kidney transplantation (ABO-iLKT) has increased the possibilities for kidney transplantation in patients with end stage renal disease. Due to advancements in immunosuppressive agents and the identification of immunological conditions following ABO-iLKT, this transplantation technique has achieved the same success rate as ABO-compatible LKT. However, some patients continue to generate anti-blood type antibodies, despite conventional immunosuppressant treatment.Case presentationA 60-year-old man was referred to our hospital for kidney transplantation. The proposed transplant was ABO incompatible, from a donor with blood-type A to a recipient with blood-type O. The recipient’s anti-A blood-type IgG antibody titer was measured at 4096-fold dilution. Following desensitization therapy, including mycophenolate mofetil (MMF) 750 mg/day for 3 months, intravenous Rituximab 200 mg, and two sessions of double filtration plasmapheresis, the anti-A blood-type IgG antibody titer decreased to only 516-fold dilution and did not meet our target of less than 128-fold dilution. MMF was thus continued for an additional 4 months and four additional sessions of plasmapheresis were undertaken. Following these interventions, antibody titers decreased to 128-fold dilution and ABO-iLKT was performed. Following transplant, antibody-mediated rejection was not observed and renal function was preserved. However, a post-operative renal biopsy 1.5 months later showed evidence of T-cell-mediated rejection IB. The patient was treated with steroids, with no increase in serum creatinine.ConclusionOur findings suggest that the long-term single MMF desensitization therapy could be a suitable option for ABO-iLKT with high refractory and rebound anti-blood type antibody. Further studies are required to establish the optimal immunosuppression regimen to control B cell- mediated immunity in ABO-iLKT.

A novel method for ABO-incompatible heart transplantation

Since 1996, ABO-incompatible heart transplantation has been undertaken by performing whole-body plasma exchange to remove isohemagglutinins using the cardiopulmonary bypass (CPB) circuit at the time of transplantation. This requires large volumes of donated blood and blood products, causes hemodynamic instability during the exchange transfusion, and limits practical use to small children. We sought to determine the efficacy of anti-A/B immunoadsorption within the CPB circuit on removal of isohemagglutinins in an ex vivo setting before its use clinically. An anti-A/B immunoadsorption column was placed into a CPB circuit mimicking a typical ABO-incompatible transplant patient, which had been primed with type O whole human blood. Samples were taken for determination of isohemagglutinin titers following each plasma volume pass through the anti-A/B immunoadsorption column. There was a linear decrease of at least 1 dilution seen in both anti-A and anti-B IgG and IgM antibodies with each plasma volume pass through the column. This predictable removal allowed the formulation of selection criteria for ABO-incompatible heart transplantation given the reciprocal of titer and patient weight. This degree of predictability allowed us to use it successfully in the clinical setting, reducing antibodies to an undetectable level during ABO-incompatible heart transplantation. The incorporation of an anti-A/B immunoadsorption column into the extracorporeal circuit reduces allogeneic blood product requirement for ABO-incompatible heart transplantation, while providing efficacious removal of anti-A and anti-B isohemagglutinins. This can be undertaken within the time period of CPB before graft reperfusion and expands the potential recipient pool to larger patients with higher isohemagglutinin titers.

Dangerous universal donors: the reality of the Hemocentro in Belo Horizonte, Minas Gerais.

BackgroundThe term dangerous universal blood donor refers to potential agglutination of the erythrocytes of non-O recipients due to plasma of an O blood group donor, which contains high titers of anti-A and/or anti-B hemagglutinins. Thus, prior titration of anti-A and anti-B hemagglutinins is recommended to prevent transfusion reactions.ObjectiveThe aim of this study was to estimate the frequency of dangerous universal donors in the blood bank of Belo Horizonte (Fundação Central de Imuno-Hematologia – Fundação Hemominas – Minas Gerais) by determining the titers of anti-A and anti-B hemagglutinins in O blood group donors.MethodA total of 400 O blood group donors were randomly selected, from March 2014 to January 2015. The titers of anti-A and anti-B hemagglutinins (IgM and IgG classes) were obtained using the tube titration technique. Dangerous donors were those whose titers of anti-A or anti-B IgM were ≥128 and/or the titers of anti-A or anti-B IgG were ≥256. Donors were characterized according to gender, age and ethnicity. The hemagglutinins were characterized by specificity (anti-A and anti-B) and antibody class (IgG and IgM).ResultsAlmost one-third (30.5%) of the O blood group donors were universal dangerous. The frequency among women was higher than that of men (p-value = 0.019; odds ratio: 1.66; 95% confidence interval: 1.08–2.56) and among young donors (18–29 years old) it was higher than for donors between 49 and 59 years old (p-value = 0.015; odds ratio: 3.05; 95% confidence interval: 1.22–7.69). There was no significant association between dangerous universal donors and ethnicity, agglutinin specificity or antibody class.ConclusionEspecially platelet concentrates obtained by apheresis (that contain a substantial volume of plasma), coming from dangerous universal donors should be transfused in isogroup recipients whenever possible in order to prevent the occurrence of transfusion reactions.

First Report on the OPTN National Variance: Allocation of A2 /A2 B Deceased Donor Kidneys to Blood Group B Increases Minority Transplantation.

In 2002, the Organ Procurement and Transplantation Network (OPTN) Minority Affairs Committee (MAC) implemented a national, prospective, "variance of practice" to allow deceased donor, ABO blood group incompatible, A2 antigen, kidney transplantation into blood group B recipients; outcomes of this cohort were compared to ABO compatible recipients. The goal of the variance was to increase the number of transplants to B candidates without negatively impacting survival or compromising system equity. Only B recipients with low anti-A IgG titers (<1:8) were eligible to receive these kidneys. Across eight participating Donation Service Areas (DSA), there were 101 A2 /A2 B to B transplants through 12/31/11, of which the majority of the recipients (61%) were ethnic minorities. At 12, 24, and 36 months, Kaplan-Meier graft survival rates for the B recipients of A2 /A2 B kidneys were 95.0%, 90.6%, and 85.4%, respectively, comparable to outcomes for B recipients of B kidneys, 92.6%, 87.9%, and 82.5%, respectively (p-value = 0.48). Five DSAs increased the proportion of B transplants during 41 months postvariance, with a lesser proportional decrease in blood group A transplants. The data support the proposition that this allocation algorithm may provide a robust mechanism to increase access of blood group B minority candidates to kidney transplantation.

Transplantation of ABO A2 Kidneys Into O Recipients: Do IgM Anti-A1 Titers Matter?

Purpose: Previous studies have shown successful kidney transplantation from ABO A2 donors into O or B recipients with low anti-A titers. To increase organ availability the new UNOS kidney allocation system (KAS) will incorporate A2 to B allocation. Criteria for A2 to O or B kidney transplants (txs) vary, from anti-A IgG titers (DTT treated serum) <1:8 (1) to anti-A IgM/G <1:16 or IgM/G > 1:8 with pre-tx plasmapharesis (2). Some report higher early graft failure with IgG titers >1:8, others found no correlation between IgG titers and acute rejection (AR) or allograft loss (1,2). The UNOS KAS does not provide anti-A titer criteria for A2 to B transplants. We report a high incidence of early AR or thrombotic microangiopathy (TMA) in A2 to O living donor (LD) transplant recipients with low IgG but high IgM/G titers. Methods: A retrospective review identified seven A2 to O LD kidney txs over 12 years at our institution. Both IgM/G and IgG (DTT treated) titers were reported. All patients (pts) received antibody induction and Tac/MMF/Pred maintenance. Results: Results are summarized in Table 1. All pts transplanted had pre-tx IgG anti-A titers <1:8. 4 pts had early allograft dysfunction with AR or TMA or both (mean 7 days post tx; range 2-16 d) with one graft loss POD 8 due to accelerated AR and cortical necrosis. Of these 4 pts, 3 had IgM/G titers >1:8 and the 4th had IgM/G=1:8. Of the three pts without rejection, one had IgM/G > 1:8.Table: No Caption available.Conclusions: Although good long term outcomes were achieved, our data show a high incidence of early AR and/or TMA and suggest caution in A2 to O kidney txs with high recipient anti-A IgM/G titers despite low IgG titers. Steps to lower anti-IgM/G pre-tx may reduce the risk of early AR or TMA. 1. Transpl 2005;80:75 2 Trans Proc 2001;33:3327 DISCLOSURES:Shaffer, D.: Grant/Research Support, Novartis, Astellas.

Selective capture of human red blood cells based on blood group using long-range surface plasmon waveguides

An optical biosensor based on long-range surface plasmon-polariton waveguides is applied to the detection of blood group antigen A on whole erythrocytes. The biosensor consists of straight gold waveguides embedded in CYTOP with an etched fluidic channel. The gold waveguides were functionalized with immunoglobulin G against blood group A (anti-A IgG) by forming a self-assembled monolayer (SAM) of 16-mercaptohexadecanoic acid (16-MHA) and then conjugating the anti-A IgG through carbodiimide chemistry. In order to demonstrate anti-A surface selectivity, solutions of O-type, B-type, A-type and AB-type red blood cells (RBCs) were sequentially injected over an anti-A functionalized waveguide. Surfaces were regenerated by lysing attached cells with distilled/deionized water (DDI H2O). The efficiency of surface regeneration with DDI H2O was very high as determined by performing six sequential binding/regeneration cycles of A RBC capture on the same anti-A surface. Also, five solutions of different A RBC concentrations, ranging from 1.14×105cells/ml to 1.83×106cells/ml, were injected over an anti-A surface to determine the limit of detection (LOD), which was found to be less than 3×105cells/ml. Finally, the response produced by a single cell bound to a waveguide was determined by relating the number of bound cells to the response produced, from which the signal-to-noise ratio for single cell detection was determined to be ~95. The waveguides are promising as simple, low-cost and compact transducers, functionalized using standard thiol-based chemistries, for the selective detection of cells.